This project is aimed at understanding the molecular basis of intracellular iron metabolism. The cis and trans elements mediating the iron-dependent alterations in abundance of ferritin and the transferrin receptor have been identified and characterized in previous years in this laboratory. Iron-responsive elements (IREs) are RNA stem-loops found in the 5' end of ferritin mRNA and the 3' end of transferrin receptor mRNA. We have cloned, expressed, and characterized an essential iron-sensing protein, the iron-responsive element binding protein (IRE_BP). The IRE-BP binds IRE's when iron levels are depleted, resulting in the inhibition of translation of ferritin mRNA and prolongation of the half-life of the transferrin receptor mRNA. The IRE- BP is 30% identical in amino acid sequence to aconitase, a mitochondrial Krebs cycle enzyme. Mitochondrial aconitase has previously been purified and crystallized and all active site residues are identical in the two proteins. Three of the active site residues are cysteines that ligate an iron-sulfur cluster that has the relatively unusual feature of containing a labile fourth iron. The IRE-BP has aconitase activity, and in vitro manipulations of iron result in changes in RNA binding. In vivo, reciprocal regulation of aconitase activity and RNA binding can be seen when the IRE-BP is over-expressed in a stable cell line. Regulation of RNA binding activity involved a transition from a [4Fe-4S] that does not bind RNS and is an active aconitase to a form that loses iron and aconitase activity. Controlled degradation of the iron-sulfur cluster reveals that the apoprotein is the physiologically relevant form of the protein in iron-depleted cells. Thus, the cluster appears to be undergoing constant disassembly, and reassembly depends on the presence of sufficient iron pools. The study of regulated iron uptake in the genetically manipulable simple eukaryote Saccharomyces cerevesiae has led to the identification of two molecularly distinct components of the cellular iron uptake system: a ferric reductase and a ferrous transporter. The two components are coordinately regulated by cellular iron availability. Continued characterization of this system is expected to provide an example of the regulatory circuit with important similarities to the human system. Identification of new genes involved in iron uptake in yeast may facilitate the identification of their human homologues.